Optimization of Osteopontin Recombinant Protein as a Candidate Supplementation for Semen Preservation

The recombinant protein of heterologous proteins in Escherichia coli strains K12 has various and different systems tested demands a detailed insight into the multiple factors affecting encoded protein. One crucial is acceptable quality DNA copies inserted inside bacteria. Firstly, amplification procedure needed to be performed well; thus, designing primer selecting optimum annealing temperature are focus indicators this study. This study obtained reference gene from NCBI data bank with Reference Sequence: NM_174187.2. Two types primers (SPP1FSPP1R OPN1F - OPN1R) targeted bands were designed selected after being reconstructed using software. Online software such as addgene.org also used identify right restriction site. distinguished PCR system amplify each primer. result revealed best at 65ºC successfully amplified 820 bp band. phenomenon not following theory blue-white screening empty plasmid control, where single colony grows on media. Competent cells plasmids should still able expand LB-Amp agar media because presence these capable providing resistance antibiotics (in case, ampicillin). discrepancy thought have been caused by improper insertion so that did enter competent cell.